Effects of PLGA nanofibre on osteoarthritic chondrocytes

Chondrocytes obtained from osteoarthritis (OA) joints has been recognized as an abnormal cell; however, it’s proven to have potential in supporting cartilage regeneration. We have isolated chondrocytes from OA joints (OAC) and expanded chondrocytes growth medium (CGM). The growth kinetic, immunophenotyping and cell multilineage differentiation were analyzed to confirm the OAC stemness. The optimal condition to developed PLGA nanofibre with ratio 50:50 were 20% concentration of PLGA, flow rate with 0.3 mL/h, 10 kv voltage and 10 cm distance from nozzle to the collector. The toxicity level, scanning electron microscopy (SEM) and q-PCR analysis was performed in the present study. OAC fulfills the minimal criteria to be known to have stem cell as the cell easily adheres to the culture plate, shows high expression (≥95%) for CD13, CD29, CD44, CD73 and CD90 and less expression (≤2%) for CD45 and HLA-DR and potentially induced to mesodermal multilineage, which is osteocytes, adipocytes and chondrocytes. Toxicity test showed no adverse effect of PLGA towards the cell. Based on the cell-PLGA nanofibre interaction, difference in fibre size will influence the proliferation of the cell. Nanofibres with 100 nm in size showed high proliferation of OAC and better gene and protein expression compared to monolayer culture. Thus, we concluded that OAC has the potential to be used in cartilage regeneration based on the presence of stem cell markers as similar to the human bone marrow. The cartilage regeneration will be more efficient if OAC cultured on 3D microenvironment as showed in the present study

Physicochemical and structural characterization of surface modified electrospun PMMA nanofibre

Although electrospun poly(methyl methacrylate) (PMMA) may mimic structural features of extracellular matrix, its highly hydrophobic nature causes reduced cell attachment. This study analysed the physicochemical and structural changes of the surface modified PMMA nanofiber. The electrospun PMMA nanofibers (PM) were surface-treated as follows: PM alone, collagen coated-PM (PM-C), UV-irradiated PM (PM-UV), collagen coated UV-irradiated PM (PM-C-UV) and collagen coated-PM crosslinked with genipin (PM-C-GEN). They were subjected to scanning electron microscopy, Fourier transform infrared (FTIR), cell attachment analysis, X-ray photoelectron spectroscopy (XPS), atomic force microscopy and X-ray powder diffraction (XRD). The surface roughness was lower in PM-C-UV group compared to others. Based on FTIR results, all expected functional group were present in all groups. XPS result showed that there are changes in the mass concentration of UV-treated surfaces and in the collagen coated surfaces. All PM groups showed amorphous nature through XRD. UV irradiation and collagen coating were shown to increase PM’s functional groups and modify its surface, which contributed to the increased attachment of cells onto the inert PM scaffold. As conclusion, collagen coated UV irradiated PMMA provided a better surface for cell to attach hence are suitable to be used further as scaffold for in vitro model

Comparative study on cartilage tissue collected from less- and severely-affected region of osteoarthritic knee

Culture expanded chondrocytes isolated from non-load bearing region of osteoarthritic (OA) joint has been used to construct tissue engineered cartilage for treatment purposes. The aim of the study was to compare the histological properties of the cartilage tissue and morphological properties of the chondrocytes isolated from less and severely affected OA knee. Human articular cartilage was obtained as redundant tissue from consented patients with late-stage OA undergoing total knee replacement surgery at Universiti Kebangsaan Malaysia Medical Centre (UKMMC). Articular cartilage was graded according to Dougados and Osteoarthritis Research Society International (OARSI) classification. Articular cartilage was classified into less affected (LA; Grade 0-1) and severely affected (SA; Grade 2-3). Cartilage tissue from less and severely affected region was stained with Safranin O staining. Isolated chondrocytes from each group were cultured until passage 4 (P4). Their growth patterns, cell areas, and circularity were compared. LA-cartilage tissue shows uniform spread of safranin O staining indicating intact extracellular matrix (ECM) component. However, SA-cartilage shows significant reduction and unstable staining due to its degraded ECM. LA-chondrocytes showed an aggregated growth compared to SA-chondrocyte that remains monolayer. Moreover, LA-chondrocytes have significantly higher cell area with wider spreading at passage 0 and 4 compared to SA-chondrocytes. It was also found that chondrocyte circularity increased with passage, and circularity of LAchondrocytes was significantly higher than that of the SA-chondrocytes at passage 3. This study demonstrated the considerable difference in the cellular properties for less and severely affected chondrocytes and implication of these differences in cell-based therapy needed to be explored

Effect of dermal fibroblast conditioned medium on keratinocytes irrespective of age group

Skin aging causes delayed re-epithelialisation and impaired wound healing. Thus, supplementation of wound healing mediators and extracellular matrix (ECM) components may be a potential treatment strategy for age-related impaired wound healing. Fibroblasts secrete wound-healing factors and can be collected from used medium, i.e., dermal fibroblast conditioned medium (DFCM). In this study, we elucidated the effect of DFCM on the in vitro wound healing of keratinocytes isolated from different age groups (≥18–35, 36–54, ≥55 years) via cell attachment, growth rate, and wound healing rate assays. The DFCM was prepared by culturing confluent fibroblasts with serum-free keratinocytespecific (DFCM-KM) and fibroblast-specific (DFCM-FM) medium. The cell attachment efficiency decreased with the increase of age. However, keratinocyte attachment was enhanced in the DFCM-KM group, where it was 1.24, 1.27, and 1.32 times higher of cells concentration for the ≥18–35-, 36–54-, and ≥55-year age groups, respectively, as compared to the control group. The keratinocytes from each age group demonstrated a similar growth profile for all culture conditions, where the DFCM-KM group exhibited a comparable growth rate with the control group whilst the DFCM-FM group exhibited a significantly lower growth rate compared to the other groups. In contrast, the DFCM-FM group demonstrated a significantly higher healing rate in all age groups as compared to the DFCM-KM and control groups. However, there was no significant difference between the healing rates of the DFCM-KM and control groups. In conclusion, DFCM-KM enhanced keratinocyte attachment while DFCM-FM enhanced the keratinocyte healing rate irrespective of donor age, which indicated the potential application of DFCM in wound healing in aged skin

3D Printed Bioscaffolds for Developing Tissue-Engineered Constructs

Tissue engineering techniques enable the fabrication of tissue substitutes integrating cells, biomaterials, and bioactive compounds to replace or repair damaged or diseased tissues. Despite the early success, current technology is unable to fabricate reproducible tissue-engineered constructs with the structural and functional similarity of the native tissue. The recent development of 3D printing technology empowers the opportunities of developing biofunctional complex tissue substitutes via layer-by-layer fabrication of cell(s), biomaterial(s), and bioactive compound(s) in precision. In this chapter, the current development of fabricating tissue-engineered constructs using 3D bioprinting technology for potential biomedical applications such as tissue replacement therapy, personalized therapy, and in vitro 3D modeling for drug discovery will be discussed. The current challenges, limitations, and role of stakeholders to grasp the future success also will be highlighted

In vitro and In vivo wound healing studies of methanolic fraction of Centella asiatica extract

Ethnopharmacological relevance: Asiaticoside is claimed as a bioactive compound capable of wound healing. In order to ensure that the pharmacological activity of the extract is traceable and measurable, the present study attempted to evaluate the bioactivity of rich fractionated extract of asiaticoside. Aim of the study: The current study evaluates the wound healing efficacy via in vitro scratch assay and in vivo circular wound excision model. Materials and methods: The ethanol extract was fractionated into seven fractions via vacuum liquid chromatography. The compound of interest in the fractions was qualitatively identified using thin layer chromatography and the positive fraction containing asiaticoside was further quantified using reverse-phase HPLC. The asiaticoside-rich fraction was subjected to (i) colorimetric MTT (methylthiazoltetrazolium) cytotoxicity assay following incubation with human dermal fibroblast (HDF) and human dermal keratinocyte (HaCaT); (ii) in vitro 12-well plate scratch assay (using HDF and HaCaT cells) and (iii) topically apply (40%, 10% and 2.5%, w/w) on in vivo circular wound excision of rabbits. Data on wound contraction, epithelisation period, hydroxyproline content and histophatological analysis was collected from in vivo study. Results: The results showed that the methanol fraction of the extract contained about 2.4% asiaticoside. Based on the results of colorimetric MTT (methylthiazoltetrazolium) cytotoxicity assay, both HDF and HaCaT showed significant stimulation upon application of the methanolic fraction of extract at concentrations of 100 μg/mL and 0.19 μg/mL. The methanol fraction showed almost no toxicity effect at the concentrations tested since their IC50 could not be determined in concentrations ranging from 100 μg/mL to 0.19 μg/mL. Since all the concentrations tested allowed for more than 90% cell viability, the concentrations chosen for the scratch assay were randomly chosen and designated as highest (100 μg/mL), medium (6 μg/mL) and lowest (0.2 μg/mL) concentrations. In the scratch assay, methanol fraction of extract with concentration of 0.2 μg/mL and 100 μg/mL showed significant effect on HDF and HaCaT compared to the positive control (p < 0.05). In vivo, it was shown that the methanol fraction of the extract induced collagen synthesis. Histopathology data also concluded that dose-dependent effect of the tested extract as a wound healer was present. Conclusions: Taken together, recent findings suggest that methanol fraction of C. asiatica demonstrated remarkable polyvalent activity, and thus has potential as an effective wound healer. In conclusion, the claim of the presence of wound healing properties in C. asiatica had been well supported based on the results obtained in this study

Exploring the potential of dermal fibroblast conditioned medium on skin wound healing and anti-ageing

Skin ageing is associated with a decrease in collagen and delayed wound healing. The previous study has shown that supplementation of growth factors can enhance the production of extracellular matrix (ECM) and efficiency of wound healing. This study aimed to produce a Dermal Fibroblast Conditioned Medium (DFCM) and evaluate its potential on in vitro skin wound healing and anti-ageing properties. DFCM were obtained by culturing confluent human dermal fibroblasts (n=3) in keratinocyte-specific medium (DFCM-KM), fibroblast-specific medium (DFCM-FM) and fibroblast-specific medium with growth supplements (DFCM-GM). Protein concentration was determined by Bicinchoninic Acid (BCA) assay. To evaluate the wound healing and anti-ageing properties, human dermal fibroblasts were supplemented with three different DFCM. Fibroblasts cultured with F12:DMEM+10%FBS was used as a control. The growth rate of fibroblasts was evaluated by culturing cells until day 4. Migration rate was evaluated at 12 h after seeding. In vitro healing rate was evaluated via scratch assay. Anti-ageing potential of DFCM was evaluated via RT2 Profiler PCR Array. Concentration of total protein was found to be significantly higher in DFCM-GM (2914.79±150.67mg/mL) compared to DFCM-KM and DFCM-FM. Out of the tested 12 anti-ageing genes, fibroblasts supplemented with DFCM-GM demonstrated a significantly higher expression of WRN compared to other conditions. In conclusion, DFCM-GM contains more proteins and found superior in modulating wound healing and anti-ageing properties

Laminin-Coated Poly(Methyl Methacrylate) (PMMA) Nanofiber Scaffold Facilitates the Enrichment of Skeletal Muscle Myoblast Population

Myoblasts, the contractile cells of skeletal muscle, have been invaluable for fundamental studies of muscle development and clinical applications for muscle loss. A major limitation to the myoblast-based therapeutic approach is contamination with non-contractile fibroblasts, which overgrow during cell expansion. To overcome these limitations, this study was carried out to establish a 3D culture environment using nanofiber scaffolds to enrich the myoblast population during construct formation. Poly(methyl methacrylate) (PMMA) nanofiber (PM) scaffolds were fabricated using electrospinning techniques and coated with extracellular matrix (ECM) proteins, such as collagen or laminin, in the presence or absence of genipin. A mixed population of myoblasts and fibroblasts was isolated from human skeletal muscle tissues and cultured on plain surfaces, as well as coated and non-coated PM scaffolds. PMMA can produce smooth fibers with an average diameter of 360 ± 50 nm. Adsorption of collagen and laminin on PM scaffolds is significantly enhanced in the presence of genipin, which introduces roughness to the nanofiber surface without affecting fiber diameter and mechanical properties. It was also demonstrated that laminin-coated PM scaffolds significantly enhance myoblast proliferation (0.0081 ± 0.0007 h−1) and migration (0.26 ± 0.04 μm/min), while collagen-coated PM scaffolds favors fibroblasts proliferation (0.0097 ± 0.0009 h−1) and migration (0.23 ± 0.03 μm/min). Consequently, the myoblast population was enriched on laminin-coated PM scaffolds throughout the culture process. Therefore, laminin coating of nanofiber scaffolds could be a potential scaffold for the development of a tissue-engineered muscle substitute

Epithelial to mesenchymal transition and eepithelialisation in wound healing: a review of comparison

Skin wound healing is a complex physiological event, involving many cellular and molecular components. The event of wound healing is the coordinated overlap of a number of distinct phases, namely haemostasis, inflammatory, proliferative and remodelling. The molecular events surrounding wound healing, particularly the reepithelialisation, has been reported to be similar to the epithelial to mesenchymal transition (EMT). In this review, the mechanism between epithelialisation and EMT were compared. Both are characterised by the loss of epithelial integrity and increased motility. In terms of the signalling kinases, Smad and mitogen-activated protein kinase (MAPK) has been reported to be involved in both reepithelialisation and EMT. At the transcriptional level, SLUG transcription factor has been reported to be important for both reepithelialisation and EMT. Extracellular matrix proteins that have been associated with both events are collagen and laminin. Lastly, both events required the interplay between matrix metalloproteinases (MMPs) and its inhibitor. As a conclusion, both reepithelialisation and EMT shares similar signaling cascade and transcriptional regulation to exhibit decreased epithelial traits and increased motility in keratinocytes